Biotherapeutics is a fast-growth sector in the pharmaceutical market with monoclonal antibodies accounting for almost 50% of the 100 billion USD spent each year on protein therapies. Bacterial fermentation, which accounts for about 35% of all protein therapeutics manufacture, continues to be the most economical method of generating proteins that do not require eukaryotic post-translational modifications. The need for efficient and economical bacterial bioprocessing becomes even more important as products such as single chain antibodies gain traction as inexpensive alternatives to monoclonal antibody therapeutics, which are the most expensive of all drugs. The common industry bacterial strains, however, can be problematic. Unmodified strains suffer from overproduction-induced stress responses that limit stable growth and can cause cell lysis. Further, biologics, such as single-chain antibodies, are often protease-sensitive and difficult to produce in common industry strains. Scarab Genomics' unique production platform Clean Genome(R) E. coli was engineered by genomic modifications to the K-12 strain MG1655, eliminating unwanted or unnecessary DNA including mobile insertion elements and prophage. Other deletions were designed to eliminate noxious sequences that can limit their productivity in bioprocessing, and enhance recombinant protein synthesis and plasmid DNA production. The current application proposes to build on the most advanced Clean Genome strain now incorporating 69 deletions for a 20% genome reduction, by generating from this starting point, a new strain combining a low mutation rate with low protease activity. In Phase I we propose to remove protease genes and error-prone polymerase genes to construct a single strain with these properties. This strain will be tested for its capacity to produce simple proteins and plasmid DNA. In Phase II the genetic background will be further refined by deleting from the genome the 23 remaining stress-induced toxin/antitoxin genes and the quorum-sensing gene luxS. This unique stress-resistant strain will be evaluated for optimal growth and production characteristics including its ability to effectively produce single- chain antibodies and other protease-sensitive proteins. Further, it will be tested for production of plasmids with difficult-to-replicate secondary structure. Finally, to meet commercialization standards, the new strain will be compared with competing industry strains in fed-batch fermentations. When developed, the single all-purpose production strain will greatly simplify the manufacture of biologics such as single-chain antibodies and offer the pharmaceutical industry a tool by which the most difficult but high-value biologics can be made efficiently and cheaply.